The present invention relates to a novel membrane-bound metalloprotease, soluble secreted form protein thereof, DNAs encoding the proteins, and medicaments based thereon.
Infiltration and metastasis of cancer cells accompanies destruction of related tissues. It has been shown that a variety of proteases are involved in the process. Therefore, inhibitors of metalloproteases are potential candidate compounds for carcinostatics and anti-metastatic agents.
On the other hand, there are a group of disorders characterized by abnormal structural alteration of proteins, which are caused by protease-resistant deposits of proteins of beta-sheet structure formed as a result of alteration in the folding process of proteins, such as spongiform encephalopathy, Alzheimer""s disease, familial amyloidosis, sickle cell anemia, pulmonary emphysema, cirrhosis, platelet thrombosis, vascular edema and the like. They are called xe2x80x9cconformational diseasesxe2x80x9d. It is known that in various conformational diseases, there are many cases in which proteins of the proteolytic system such as protease inhibitors undergo conformational changes and accumulate. Proteases with relatively low substrate specificity and having ability to decompose denatured proteins at their early stages, therefore, would have potential to become agents for prophylaxis or treatment of those diseases.
A wide variety of biologically active peptide hormones, regulatory peptides and neuropeptides have been shown to be proteolytically activated or inactivated by members of zinc metalloproteases [Hooper, N. M., FEBS Lett., 354:1-6(1994)]. One such class of zinc metalloproteases, represented by neutral endopeptidase 24.11 (NEP) and endothelin-converting enzyme (ECE), has recently been highlighted because of their implications in some disease states and should thus provide plausible therapeutic targets for certain diseases [Yanagisawa, M., Circulation, 89:1320-1322(1994); Turner, A. J. et al., Biochem. Pharmacol., 51:91-102(1996); Turner, A. J. et al., FASEB J., 11:355-364(1997)]. In mammals, six members of this metalloprotease family have been identified; NEP; Kell blood group antigen (KELL); ECE-1 and ECE-2; PEX, which has been associated with X-linked hypophosphatemic rickets; and a recently identified peptidase, XCE. All these members are type II membrane proteins containing a highly conserved consensus sequence of a zinc-binding motif, HEXXH (where X represents any amino acid), in their extracellular C-terminal domain. Despite the apparent structural similarity among the members of this family, a large diversity of physiological functions exists.
NEP, which is especially abundant in kidney and brain, is also expressed in various tissues as an ectoenzyme that can degrade many circulating small peptide mediators, such as enkephalins, atrial natriuretic peptide (ANP), tachykinins, and endothelins (ETs) [Roques, B. P. et al., Pharmacol. Rev., 45:87-146(1993)]. NEP is also known as the common acute lymphoblastic leukemia antigen, and its presence on leukemic cells has been associated with a better prognosis. Although the physiological substrates of NEP are still unknown, targeted disruption of the NEP gene in mice caused a dramatic sensitivity to endotoxin shock, suggesting that NEP may provide an unexpected protective role against endotoxin shock [Lu, B. et al., J. Exp. Med., 181:2271-2275(1995)]. Moreover, in vivo pharmacological inhibition of NEP has led to a decrease in blood pressure, and NEP-deficient mice were noted to have lower mean blood pressure levels than wild-type littermates, thus indicating that NEP may also play an important role in blood pressure regulation [Lu, B. et al., Nat. Med., 3:901-907(1997)].
ECE, another well characterized member of this metalloprotease family, is involved in the regulation of vascular tone, as well as in the development of some sets of neural crest cells [Turner, A. J. et al., Biochem. Pharmacol., 51:91-102(1996); Turner, A. J. et al., FASEB J., 11:355-364(1997)]. It converts the inactive ET precursors (big ETs) into biologically active ETs via a specific cleavage at Trp21-VAl/Ile22 [Yanagisawa, M. et al, Nature, 332:411-415(1988); Xu, D. et al., Cell, 78: 473-485(1994)]. ECE constitutes a potential regulatory site for the production of the active peptide. Two isozymes of ECE, ECE-1 and ECE-2, have been molecularly identified and make up a subfamily within this group of type II membrane-bound metalloproteases [Xu, D. et al., Cell, 78:473-485(1994); Emoto, N. et al., J. Biol. Chem., 270:15262-15268(1995)]. Both enzymes have been shown to cleave big ET-1 to produce ET-1 with a similar overall profile of inhibitor sensitivity in vitro as well as in transfected cells. However, ECE-1 and ECE-2 exhibit the following striking differences: (i) ECE-1 cleaves big ETs in neutral pH, whereas ECE-2 functions in an acidic pH range, (ii) the sensitivity of ECE-1 to phosphoramidon is 250-fold lower than that of ECE-2 and (iii) ECE-1 is abundantly expressed in endothelial cells and other cell types known to produce mature ET-1, whereas ECE-2 mRNA is detected in neural tissues including the cerebral cortex, the cerebellum, and the adrenal medulla. Targeted disruption of the ECE-1 gene in mice revealed that ECE-1 is the physiologically relevant enzyme needed to produce active ET-1 [Yanagisawa, H. et al., Development, 125:825-836(1998)]. The physiological function of ECE-2 has not yet been elucidated.
The physiological substrates of the three other mammalian peptidases, KELL, PEX, and XCE, are still unknown. KELL, expressed on human red cells and other cell types, carries the epitopes for the KELL minor blood group antigen. Although the KELL blood group antigen is clinically important, its actual protease activity has yet to be described. The PEX gene was identified by positional cloning as a candidate gene for X-linked hypophosphatemic rickets, a dominant disorder characterized by impaired phosphate uptake in the kidney. XCE was recently isolated by screening EST date base with the ECE-1 sequence. No protease activity for XCE has been detected, hence, its physiological significance is unknown.
Recent gene targeting studies of ECE revealed that ECE-1 is a bona fide activating protease for big ET-1 and big ET-3 at specific developmental stages [Yanagisawa, H. et al., Development, 125:825-836(1998)]. However, despite the absence of ECE-1 (which resulted in craniofacial and cardiovascular defects), a significant amount of mature ET-1 peptide was still found in ECE-1xe2x88x92/xe2x88x92 embryos, suggesting that other proteases can activate ET-1.
Upon the above-described background, the present inventors attempted to find out enzymes structurally relating to the metalloprotease family. The present inventors, as a result, successfully isolated a novel enzyme, termed soluble secreted endopeptidase (SEP), and its membrane bound form SEPxcex94, which lacks part of its amino acids, by degenerate PCR using cDNA prepared from ECE-1xe2x88x92/xe2x88x92 embryos as template. SEP and SEPxcex94 polypeptides are identical with each other except for the 23 amino acids characteristic of SEP. Its DNA sequence predicts that SEP is a type II membrane-bound metalloprotease structurally related to NEP, ECE-1, and ECE-2. Transfection of the SEP cDNA into Chinese hamster ovary (CHO) cells resulted in the occurrence of SEP protein not only in the membrane fraction of the cells but also in the supernatant, suggesting that these cells release soluble forms of the enzyme through proper secretory machinery. Enzymological analysis of the recombinant soluble SEP protein revealed that SEP hydrolyzes a variety of peptides, which are known as substrates of NEP and/or ECE, including big ET-1, ET-1, angiotensin 1, ANP, bradykinin, and substance P. This suggest that SEP is likely a novel member of this metalloprotease family and may be involved in the metabolism of biologically active peptides.
Thus, the present invention provides proteins (SEP and SEPxcex94, respectively) and salts thereof having amino acid sequences set forth under SEQ ID NO:1 and NO:2, respectively, in the Sequence Listing.
The present invention further provides metalloprotease proteins and salts thereof having a neutral optimal pH and having amino acid sequences substantially the same as the amino acid sequence set forth under SEQ ID NO:1 or NO:2 in the Sequence Listing, and which has an activity to hydrolyze endothelin 1, atrial natriuretic peptide and angiotensin I and are inhibited by 1,10-phenanthroline, phosphoramidon and thiorphan.
The present invention further provides DNAs having nucleotide sequences encoding the above-identified proteins.
The present invention further provides, in particular, DNA having the coding region nucleotide sequences of SEQ ID NO:3 or NO:4 in the Sequence Listing.
The present invention further provides DNAs having nucleotide sequences with one or more nucleotides deleted, substituted, inserted or added relative to the coding region nucleotide of SEQ ID NO:3 or NO:4 in the Sequence Listing and encoding proteins which are substantially the same as the protein encoded by the nucleotide sequence set forth under SEQ ID NO:3 or NO:4 in the Sequence Listing.
The present invention further provides DNAs comprising a nucleotide sequence which hybridize under a high-stringent condition with the DNAs having the coding region nucleotide sequences of SEQ ID NO:3 or NO:4 in the Sequence Listing.
The present invention further provides expression vectors in which on of the above-identified DNAs is incorporated.
The present invention further provides transformant cells which carry one of the above-identified vectors.
The present invention further provides a method for producing proteins or salts thereof which comprises introducing into a host cell a recombinant vector in which one of the above-identified DNAs is incorporated to form a transformant, culturing the transformant to produce a protein encoded by the one of the above DNAs, and collecting the protein.
The present invention further provide an antibody directed to the above-identified protein or peptide.
The present invention further provides pharmaceutical compositions for treatment or prophylaxis of a disease selected from the group consisting of spongiform encephalopathy, Alzheimer""s disease, familial amyloidosis, sickle cell anemia, pulmonary emphysema, cirrhosis, platelet thrombosis and vascular edema, which compositions comprise one of the above-identified proteins or a salt thereof.
The present invention further provides a screening kit for selection of carcinostatics and anti-metastatic agents, said kit comprising one of the above proteins, a substrate for protease activity of the protein and a buffering agent which are enclosed separately from each other, with which kit screening is conducted based on the measurement of inhibition of protease activity of the protein by given candidate compounds.
The present invention further provides use of one of the above proteins for the manufacture of a medicament for treatment or prophylaxis of a disease selected from the group consisting of spongiform encephalopathy, Alzheimer""s disease, familial amyloidosis, sickle cell, anemia, pulmonary emphysema, cirrhosis, platelet thrombosis and vascular edema.
The present invention further provides use of one of the above proteins in combination with a substrate for protease activity of the protein for the manufacture of a screening kit for selection of carcinostatics and anti-metastatic agents, with which kit screening is conducted based on the measurement of inhibition of protease activity of the protein by given candidate compounds